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Biological sex and age influence GS-9620 activity ex vivo

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Abstract

Toll-like receptors (TLRs) are being explored to enhance immunity in HIV cure strategies. The TLR7 agonist GS-9620 promotes immune activation, reactivates latent HIV, and delays viral rebound in some people with HIV. Previous work has shown that biological sex influences TLR7 signaling. This study examined the interplay between biological sex, age, and the sex hormones 17β-estradiol, progesterone, and testosterone on GS-9620’s ability to promote cytokine secretion and activate CD4, CD8, and NK cells ex vivo. Interestingly, sex hormones had no effect on GS-9620-mediated immune activation or cytokine induction. However, we found that GS-9620 activity was influenced by age only in female donors. Further, we found that GS-9620-mediated CD4 T cell activation was positively correlated with the induction of IFN-γ and IL-12, while CD4 T cell activation and IL-12 production were negatively correlated with age. Additionally, CD8 T cell activation was positively correlated with IFN-γ production. Mechanistically, IFN-γ was sufficient to promote higher immune activation of both CD4 and CD8 T cells in female versus male donors. In conclusion, biological sex and age, but not sex hormones, influence GS-9620-mediated immune activation. Understanding these factors will help design and evaluate future clinical trials using GS-9620 for an HIV cure.

Authors

Carissa S. Holmberg, Callie Levinger, Adam R. Ward, Alberto Bosque

Tumor suppressors in Sox2-mediated lung cancers promote distinct cell intrinsic and immunologic remodeling

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Abstract

Non-small cell lung cancer (NSCLC) largely consists of lung squamous (LUSC) and lung adenocarcinoma (LUAD). Alterations in the TRP53 and PTEN tumor suppressors are common in both subtypes, but their relationship with SOX2 is poorly understood. We deleted Trp53 or Pten in a C57BL/6J-Sox2hi;Nkx2-1-/-;Lkb1-/- (SNL) genetic background and generated a highly metastatic LUSC cell line (LN2A; derived from a Sox2hi mouse model, followed by Trp53, Pten, and Cdkn2a deletion). Histologic and single-cell RNAseq analyses corroborated that SNL mice developed mixed tumors with both LUAD and LUSC histopathology while SNL-Trp53 and SNL-Pten mice developed LUAD and LN2A tumors retained LUSC morphology. Compared with SNL mice, additional loss of Trp53 or Pten resulted in significantly reduced survival, increased tumor burden and altered tumor mucin composition. We identified a sub-cluster of CD38+ tumor-associated inflammatory monocytes in the LN2A model that significantly enriched for activation of the classical and alternative complement pathways. Complement Factor B (CFB) is associated with poor survival in LUSC patients, and we observed the LN2A model had significantly improved survival on a Cfb-/- background. Our findings demonstrate a cooperative role of Trp53 and Pten tumor suppressors in Sox2-mediated NSCLC tumor progression, mucin production, and remodeling of the immune tumor microenvironment.

Authors

Nisitha Sengottuvel, Kristina M. Whately, Jennifer L Modliszewski, Rani S. Sellers, William D. Green, Weida Gong, Allison T. Woods, Eric W. Livingston, Katerina D. Fagan-Solis, Gabrielle Cannon, Lincy Edatt, Hong Yuan, Aaron C. Chack, Yazmin Sanchez, Katherine Zhou, Alyaa Dawoud, Jarred M. Green, Virginia Godfrey, J Justin Milner, Gaorav P. Gupta, Chad V. Pecot

Imlunestrant a next-generation oral SERD overcomes ESR1 mutant resistance in estrogen receptor-positive breast cancer

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Abstract

Estrogen receptor alpha (ER) is a critical driver of tumorigenesis and tumor progression in most breast cancers. Endocrine therapies (ET) targeting ER are central to treating hormone receptor-positive breast cancer (BC), but resistance poses a clinical challenge. Some resistance mechanisms, particularly those involving estrogen-independent activity such as the ESR1 mutations, rely on ER signaling, supporting the need for next-generation ET. We investigated the preclinical efficacy of imlunestrant, an oral selective ER degrader, in ER-positive BC pre-clinical models, including models harboring the Y537S ESR1 mutation, an activating mutation. Imlunestrant demonstrated antagonistic activity and effective degradation of both wild-type and mutant ER, resulting in cell growth suppression. In vivo, imlunestrant outperformed fulvestrant leading to tumor regression in a patient derived xenograft harboring the Y537S ESR1 mutation. Cyclic mutiplexed immunofluorescence and transcriptomic analysis revealed enhanced cell cycle arrest and downregulation of estrogen-responsive genes with imlunestrant treatment. Additionally, a genome wide CRISPR knock-out screen identified several vulnerabilities that were either persistent or gained after imlunestrant treatment, providing a rationale for future studies of combination treatments with imlunestrant. Collectively, these results highlight the on-target and selective activity of imlunestrant, which can circumvent resistance engendered by the Y537S ESR1 mutation.

Authors

Shira Sherman, Zachary M. Sandusky, Douglas Russo, David Zak, Agostina Nardone, Delia Friel, Francisco Hermida-Prado, Capucine Heraud, Genevra Kuziel, Ana Verma, Giorgio Gaglia, Sheheryar Kabraji, Quang-De Nguyen, Sandro Santagata, Sean W. Fanning, Rinath Jeselsohn

Remdesivir post-exposure prophylaxis limits measles-induced “immune amnesia” and measles antibody responses in macaques

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Abstract

Measles remains one of the most important causes of worldwide morbidity and mortality in children. Measles virus (MeV) replicates extensively in lymphoid tissue and most deaths are due to other infectious diseases associated with MeV-induced loss of circulating antibodies to other pathogens. To determine whether remdesivir, a broad-spectrum direct-acting antiviral, affects MeV-induced loss of antibody to other pathogens, we expanded the VirScan technology to detect antibody to both human and macaque pathogens. We measured the antibody reactivity to MeV and non-MeV viral peptides using plasma from MeV-infected macaques that received remdesivir either as post-exposure prophylaxis (d3-14, PEP) or as late treatment (d11-22, LT) in comparison with macaques that were not treated. Remdesivir PEP, but not LT, limited the loss of antibody to non-MeV pathogens. Remdesivir PEP also limited the antibody response to MeV with a decrease in both the magnitude and breadth of the epitopes recognized. LT had little effect on the magnitude of the MeV-specific antibody response but affected the breadth of the response. Therefore, early, but not late, treatment of measles with the direct-acting antiviral remdesivir prevents the loss of antibody to other pathogens but decreases the response to MeV.

Authors

Andy Kwan Pui Chan, Liting Liu, William R. Morgenlander, Manjusha Thakar, Nadine A. Peart Akindele, Jacqueline Brockhurst, Shristi Ghimire, Maggie L. Bartlett, Kelly A. Metcalf Pate, Victor C. Chu, Meghan S. Vermillion, Danielle P. Porter, Tomas Cihlar, Michael J. Mina, H. Benjamin Larman, Diane E. Griffin

Virus-induced RGMa expression drives neurodegeneration in HTLV-1-associated myelopathy

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Abstract

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM) is a rare neurodegenerative disease with largely elusive molecular mechanisms, impeding targeted therapeutic advancements. This study aimed to identify the critical molecule responsible for neuronal damage in HAM, its source, and the regulatory mechanisms controlling its expression. Utilizing patient-derived cells and established cell lines, we discovered that HTLV-1 Tax, in conjunction with Specificity Protein 1 (Sp1), enhanced the expression of repulsive guidance molecule A (RGMa), a molecule known to contribute to neuronal damage. RGMa expression was specifically upregulated in HTLV-1-infected cells from HAM patients, particularly in those expressing HTLV-1 Tax. Furthermore, in CD4+ cells from HAM patients, the level of H3K27me3 methylation upstream of the RGMA gene locus was reduced, making RGMA more prone to constitutive expression. We demonstrated that HTLV-1-infected cells in HAM inflict neuronal damage via RGMa. Crucially, the neutralizing antibody against RGMa, unasnemab/MT-3921, effectively mitigated this damage in a dose-responsive manner, highlighting RGMa's pivotal role in neuronal damage and its potential as a therapeutic target for alleviating neuronal damage in HAM.

Authors

Natsumi Araya, Makoto Yamagishi, Makoto Nakashima, Naomi Asahara, Kazuhiro Kiyohara, Satoko Aratani, Naoko Yagishita, Erika Horibe, Izumi Ishizaki, Toshiki Watanabe, Tomoo Sato, Kaoru Uchimaru, Yoshihisa Yamano

T-cell phenotype and clonality changes in myeloma patients with short OS

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Abstract

Overall survival (OS) in multiple myeloma (MM) varies between a couple of months to more than 20 years, influenced by tumor characteristics, the tumor microenvironment (TME), and patient factors such as age and frailty. We analyzed sequential BM samples from 45 MM patients with OS < 3 years versus > 8 years using mass cytometry and bulk TCRβ sequencing. Patients with long OS demonstrated stability in the TME and T cell environments, while those with short OS had significant changes at relapse, including fewer T cells, increased Treg cells, and more activated and exhausted CD8 T cells. Notably, higher PD-1 expression in CD8 T cells at diagnosis correlated with short OS. Additionally, short-OS patients exhibited a more monoclonal T cell environment at relapse, with abundance of hyperexpanded clones. These findings reveal distinct immune cell differences between patients with short and long OS.

Authors

Alenka Djarmila Behsen, Esten Nymoen Vandsemb, Tobias Schmidt Slørdahl, Karen Dybkær, Maja Zimmer Jakobsen, Muhammad Kashif, Johan Lund, Vincent Luong, Astrid Marta Olsnes, Anders Waage, Anne Marit Sponaas, Kristine Misund

Epac1 contributes to apremilast-mediated rescue of pemphigus autoantibody-induced loss of keratinocyte adhesion

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Abstract

In the bullous autoimmune disease pemphigus vulgaris (PV), autoantibodies directed mainly against desmoglein (Dsg)1 and Dsg3 cause loss of desmosomal adhesion. We recently showed that intracellular cAMP increase by the phosphodiesterase 4 inhibitor apremilast was protective in different PV-models. Thus, we here analyzed the involvement of the cAMP effector exchange factor directly activated by cAMP (Epac)1. In Epac1-deficient mice pemphigus antibody-induced blistering was ameliorated in vivo while apremilast had no additional effect. Interestingly, augmented protein levels of Dsg1 and Dsg3 as well as increased Dsg1 mRNA levels and higher numbers of Dsg1- and Dsg3-dependent single molecule interactions were detected in keratinocytes derived from Epac1-deficient mice. This was paralleled by stronger intercellular adhesion under baseline conditions and prevention of pemphigus autoantibody-induced loss of intercellular adhesion. However, the protective effect of apremilast against loss of intercellular adhesion in response to the pathogenic Dsg3 antibody AK23 was attenuated in Epac1-deficient keratinocytes. Similarly, the Epac1 inhibitor Esi09 protected keratinocytes from pemphigus antibody-induced loss of adhesion. Mechanistically, Epac1 deficiency resulted in lack of apremilast-induced Rap1 activation and phosphorylation of Pg at S665. Taken together, these data indicate that Epac1 is involved in the regulation of baseline and cAMP-mediated stabilization of keratinocyte adhesion.

Authors

Anna M. Sigmund, Franziska C. Bayerbach, Daniela Kugelmann, Elisabeth Butz, Sina Moztarzadeh, Margarethe E.C. Schikora, Anja K.E. Horn, Mariya Y. Radeva, Sophia Engelmayer, Desalegn T. Egu, Matthias Goebeler, Enno Schmidt, Jens Waschke, Franziska Vielmuth

LRRC8 channel complexes counterbalance K_ATP channels to mediate swell-secretion coupling in mouse pancreatic β-cells

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Abstract

Insulin secretion from pancreatic β-cells is initiated by membrane potential depolarization followed by activation of voltage-gated Ca2+ channels to trigger Ca2+-mediated insulin vesicle fusion with the β-cell plasma membrane. Here, we show that β-cell swelling associated with glucose metabolism is sensed by LRRC8 channel complexes and contributes to insulin secretion. Hypertonic perfusate (360–380 mOsm) dose-dependently impairs glucose-stimulated insulin secretion by counteracting β-cell swelling. Hypotonic perfusate alone, independent of glucose stimulation or KATP channel closure, is sufficient to increase β-cell intracellular Ca2+ and trigger insulin secretion. Inhibition of sodium-potassium-chloride cotransporter-1 with bumetanide, which diminishes the intracellular Cl– concentration in β-cells and consequently reduces Cl– efflux via LRRC8 channel complexes, also significantly reduces hypotonic-stimulated insulin secretion. Finally, stimulation of insulin secretion by the glucokinase activator GKA50, which is known to induce β-cell swelling, is entirely suppressed in β-cell-targeted Lrrc8a KO islets. These data support a model wherein the LRRC8 channel complex senses β-cell swelling triggered by glucose metabolism and regulates β-cell insulin secretion through activation of LRRC8-mediated Cl– efflux.

Authors

Tarek Mohamed Abd El-Aziz, Chen Kang, Litao Xie, John D. Tranter, Sumit Patel, Rahul Chadda, Maria S. Remedi, Rajan Sah

Identification and regulation of circulating tumor TCR-matched cytotoxic CD4⁺ lymphocytes by KLRG1 in bladder cancer

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Abstract

While cytotoxic CD4+ tumor-infiltrating lymphocytes have anti-cancer activity in patients, whether these can be non-invasively monitored and how these are regulated remains obscure. By matching single cells with T cell receptors (TCR) in tumor and blood of bladder cancer patients, we identified distinct pools of tumor-matching cytotoxic CD4+ T cells in the periphery directly reflecting the predominant antigenic specificities of intratumoral CD4+ TILs. On one hand, the granzyme B (GZMB)-expressing cytotoxic CD4+ subset proliferated in blood in response to PD-1 blockade, but was separately regulated by the killer cell lectin-like receptor G1 (KLRG1) which inhibited their killing by interacting with E-cadherin. Conversely, a clonally related, granzyme K (GZMK)-expressing circulating CD4+ population demonstrated basal proliferation and a memory phenotype that may result from activation of GZMB+ cells, but was not directly mobilized by PD-1 blockade. As KLRG1 marked the majority of circulating tumor TCR-matched cytotoxic CD4+ T cells, this work nominates KLRG1 as a means to isolate them from blood and provide a window into intratumoral CD4+ recognition, as well as a putative regulatory receptor to mobilize the cytolytic GZMB+ subset for therapeutic benefit. Our findings also underscore ontogenic relationships of GZMB- and GZMK-expressing populations and the distinct cues that regulate their activity.

Authors

Serena S. Kwek, Hai Yang, Tony Li, Arielle Ilano, Eric D. Chow, Li Zhang, Hewitt Chang, Diamond Luong, Averey Lea, Matthew Clark, Alec Starzinski, Yimin Shi, Elizabeth McCarthy, Sima Porten, Maxwell V. Meng, Chun Jimmie Ye, Lawrence Fong, David Y. Oh

PAI-1 Interaction with Sortilin Related Receptor-1 is Required for Lung Fibrosis

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Abstract

Mutation studies of plasminogen activator inhibitor-1 (PAI-1) have previously implied that PAI-1 promotes lung fibrosis via a vitronectin (VTN) dependent mechanism. In the present study, employing two distinct murine fibrosis models and VTN deficient mice, we find that VTN is not required for PAI-1 to drive lung scarring. This result suggested the existence of a profibrotic interaction involving the VTN-binding site on PAI-1 with an unidentified ligand. Using an unbiased proteomic approach, we identified sortilin related receptor 1 (SorLA) as the most highly enriched PAI-1 binding partner in the fibrosing lung. Investigating the role of SorLA in pulmonary fibrosis demonstrated that deficiency of this protein protected against lung scarring in a murine model. We further found that SorLA is required for PAI-1 to promote scarring in mice, that both SorLA and PAI-1 protein levels are increased in human IPF explants, and that these proteins are associated in IPF tissue. Finally, confocal microscopy shows that expression of SorLA in CHO cells increases cell uptake of PAI-1, and these proteins colocalize in the cytoplasm. Together, these data elucidate a mechanism by which the potent profibrotic mediator PAI-1 drives lung fibrosis and implicate SorLA as a potential therapeutic target in IPF treatment.

Authors

Thomas H. Sisson, John J. Osterholzer, Lisa Leung, Venkatesha Basrur, Alexey I. Nesvizhskii, Natalya Subbotina, Mark Warnock, Daniel Torrente, Ammara Q. Virk, Sergey S. Gutor, Jeffrey C. Horowitz, Mary Migliorini, Dudley K. Strickland, Kevin K. Kim, Steven K. Huang, Daniel A. Lawrence

Lactate programs PBX1 lactylation and mesangial proliferation in lupus nephritis

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Abstract

Lupus nephritis (LN) constitutes the most common organ-threatening manifestation of systemic lupus erythematosus (SLE), with the pathological proliferation of mesangial cells (MCs) recognized as a critical factor in its pathogenesis and progression. Self-DNA-containing immune complex (DNA-IC) represents a prime pathogenic factor in SLE, yet its pathological impact on MCs remains unclear. In the present study, we elucidated the mechanism underlying the excessive proliferation of MCs following the recognition of DNA-IC in LN patients. Here, we pinpointed that the excessive proliferation of MCs was attributed to an anomalous transition from the G1 to the S phase of the cell cycle in LN patients. Mechanically, the dysfunction of P27 protein resulted in the aberrant G1-S phase transition, and the phenomenon was closely related to the ubiquitin-mediated degradation of its key transcription factor, PBX1. This degradation was regulated by lactylation of PBX1 in the site of Lys40 residue. The elevated lactylation level of PBX1 protein arisen from the up-regulation of glycolysis levels induced by DNA-IC. Accordingly, targeting lactate production in MCs of LN patients effectively alleviated renal inflammation and fibrosis progression in LN patients. Elevated lactate results in PBX1 lactylation, leading to MCs excessive proliferation and thus serving as a promising therapeutic target for LN.

Authors

Enzhuo Liu, Chenghua Weng, Chenchu Yan, Xingchen Zhu, Xinyue Li, Mengdi Liu, Zhenke Wen, Zhichun Liu

The gut microbiome enhances breast cancer immunotherapy following bariatric surgery

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Abstract

Bariatric surgery is associated with improved breast cancer (BC) outcomes, including greater immunotherapy effectiveness in a pre-clinical BC model. A potential mechanism of bariatric surgery-associated protection is the gut microbiota. Here, we demonstrate the dependency of improved immunotherapy response on the post-bariatric surgery gut microbiome via fecal microbial transplant (FMT). Response to αPD-1 immunotherapy was significantly improved following FMT from formerly obese bariatric surgery-treated mice. When stool from post-bariatric surgery patients was transplanted into recipient mice and compared to the patients’ pre-surgery transplants, post-surgery microbes significantly reduced tumor burden and doubled immunotherapy effectiveness. Microbes impact tumor burden through microbially derived metabolites, including branched chain amino acids (BCAA). Circulating BCAAs correlated significantly with natural killer T (NKT) cell content in the tumor microenvironment in donor mice after bariatric surgery and FMT recipients of donor cecal content after bariatric surgery compared to obese controls. BCAA supplementation replicated improved αPD-1 effectiveness in two BC models, supporting the role of BCAAs in increased immunotherapy effectiveness after bariatric surgery. Ex vivo exposure increased primary NKT cell expression of anti-tumor cytokines, demonstrating direct activation of NKT cells by BCAAs. Together, findings suggest that reinvigorating anti-tumor immunity may depend upon bariatric surgery-associated microbially derived metabolites, namely BCAAs.

Authors

Margaret S. Bohm, Sydney C. Joseph, Laura M. Sipe, Minjeong Kim, Cameron T. Leathem, Tahliyah S. Mims, Nathaniel B. Willis, Ubaid A. Tanveer, Joel H. Elasy, Emily W. Grey, Madeline E. Pye, Zeid T. Mustafa, Barbara Anne Harper, Logan G. McGrath, Deidre Daria, Brenda Landvoigt Schmitt, Jelissa A. Myers, Patricia Pantoja Newman, Brandt D. Pence, Marie Van der Merwe, Matthew J. Davis, Joseph F. Pierre, Liza Makowski

The cardiac METTL3-m6A pathway regulates the systemic response to western diet

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Abstract

Regulation of organismal homeostasis in response to nutrient availability is a vital physiological process that involves inter-organ communication. The role of the heart in controlling systemic metabolic health is not clear. Adopting a mouse model of diet-induced obesity, we found that the landscape of N6-methyladenosine (m6A) on cardiac mRNA is altered following high fat/high carbohydrate feeding (western diet). m6A is a critical post-transcriptional regulator of gene expression, the formation of which is catalyzed by methyltransferase-like 3 (METTL3). Through parallel unbiased approaches of nanopore sequencing, mass spectrometry, and protein array, we found regulation of circulating factors under the control of METTL3. Mice with cardiomyocyte-specific deletion of METTL3 show a systemic inability to respond to nutritional challenge, thereby mitigating the detrimental effects of western diet. Conversely, increasing cardiac METTL3 level exacerbates diet-induced body weight gain, adiposity, and glucose intolerance. Our findings position the heart at the center of systemic metabolism regulation and highlight an m6A-dependent pathway to be exploited for the battle against obesity.

Authors

Charles Rabolli, Jacob Z. Longenecker, Isabel S. Naarmann-de Vries, Joan Serrano, Jennifer M. Petrosino, George A. Kyriazis, Christoph Dieterich, Federica Accornero

METTL14 promotes intimal hyperplasia through m6A-mediated control of vascular smooth muscle dedifferentiation genes

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Abstract

Vascular smooth muscle cells (VSMCs) possess significant phenotypic plasticity, shifting between a contractile phenotype and a synthetic state for vascular repair/remodelling. Dysregulated VSMC transformation, marked by excessive proliferation and migration, primarily drives intimal hyperplasia. N6-methyladenosine (m6A), the most prevalent RNA modification in eukaryotes, plays a critical role in gene expression regulation; however, its impact on VSMC plasticity is not fully understood. This research investigates the alterations in m6A modification and its regulatory factors during VSMC phenotypic shifts and their influence on intimal hyperplasia. We demonstrate that METTL14, crucial for m6A deposition, significantly promotes VSMC dedifferentiation. METTL14 expression, initially negligible, is elevated in synthetic VSMC cultures, post-injury neointimal VSMCs, and human restenotic arteries. Reducing METTL14 in mouse primary VSMCs decreases pro-synthetic genes, suppressing their proliferation and migration. m6A-RIP-seq profiling shows key VSMC gene networks undergo altered m6A regulation in Mettl14-deficient cells. METTL14 enhances KLF4 and SERPINE1 expression through increased m6A deposition. Local METTL14 knockdown significantly curbs neointimal formation post-arterial injury, and reducing METTL14 in hyperplastic arteries halts further neointimal development. We found that METTL14 is a pivotal regulator of VSMC dedifferentiation, influencing KLF4- and SERPINE1-mediated phenotypic conversion. Inhibiting METTL14 is a viable strategy for preventing restenosis and halting restenotic occlusions.

Authors

Grace Chensee, Bob S.L. Lee, Immanuel D. Green, Jessica Tieng, Renhua Song, Natalia Pinello, Quintin Lee, Majid Mehravar, David A. Robinson, Mian Wang, Mary M. Kavurma, Jun Yu, Justin Jong Leong Wong, Renjing Liu

The potentiator ivacaftor is essential for pharmacological restoration of F508del-CFTR function and mucociliary clearance in cystic fibrosis

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Abstract

Pharmacological rescue of F508del-CFTR by the triple combination CFTR modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) leads to unprecedented clinical benefits in patients with cystic fibrosis (CF), however, previous studies in CF primary human airway epithelial cultures demonstrated that chronic treatment with the potentiator ivacaftor can render the F508del protein unstable thus limiting restoration of CFTR chloride channel function. However, quantitative studies of this unwanted effect of ivacaftor on F508del channel function including dependency on cell culture conditions remain limited and the impact of chronic ivacaftor exposure on restoration of mucociliary clearance that is impaired in patients with CF has not been studied. In patient-derived primary nasal epithelial cultures, we found that different culture conditions (UNC-ALI medium vs. PneumaCult medium) have profound effects on ETI-mediated restoration of F508del-CFTR function. Chronic treatment with ivacaftor as part of ETI triple therapy limited the rescue of F508del-CFTR chloride channel function when CF nasal epithelial cultures were grown in UNC-ALI medium, but not in PneumaCult medium. In PneumaCult medium, both chronic and acute addition of ivacaftor as part of ETI treatment led to constitutive CFTR-mediated chloride secretion in the absence of exogenous cAMP-dependent stimulation. This constitutive CFTR-mediated chloride secretion was essential to improve viscoelastic properties of the mucus layer and to restore mucociliary transport on CF nasal epithelial cultures. Furthermore, nasal potential difference measurements in patients with CF showed that ETI restored constitutive F508del-CFTR activity in vivo. These results demonstrate that ivacaftor as a component of ETI therapy is essential to restore mucociliary clearance and suggest that this effect is facilitated by its constitutive activation of F508del channels following their folding-correction in patients with CF.

Authors

Anita Balázs, Tihomir Rubil, Christine K. Wong, Jasmin Berger, Marika Drescher, Kathrin Seidel, Mirjam Stahl, Simon Y. Graeber, Marcus A. Mall

G6PC3 promotes genome maintenance and is a candidate mammary tumor suppressor

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Abstract

Mutations in genome maintenance factors drive sporadic and hereditary breast cancers. Here, we searched for potential drivers based on germline DNA analysis from a cohort consisting of early-onset breast cancer patients negative for BRCA1/BRCA2 mutations. This revealed candidate genes that subsequently were subjected to RNAi-based phenotype screens to reveal genome integrity impacts. We identified several genes with functional roles in genome maintenance, including Glucose-6-Phosphatase Catalytic Subunit 3 (G6PC3), SMC4, and CCDC108. Notably, G6PC3-deficient cells exhibited increased levels of γH2AX and micronuclei formation, along with defects in homologous recombination (HR) repair. Consistent with these observations, G6PC3 was required for the efficient recruitment of BRCA1 to sites of DNA double-strand breaks (DSBs). RNA sequencing analysis revealed that G6PC3 promotes the expression of multiple homologous recombination repair genes, including BRCA1. Through CRISPR-Select functional-genetic phenotype analysis of G6PC3 germline mutations, we identified two germline G6PC3 variants displaying partial loss-of-function. Furthermore, our study demonstrated that G6pc3 deficiency accelerates mammary tumor formation induced by Trp53 loss in mice. In conclusion, our cohort-based functional analysis has unveiled genome maintenance factors and identified G6PC3 as a potential candidate tumor suppressor in breast cancer.

Authors

Xin Li, Maria Rossing, Ana Moisés da Silva, Muthiah Bose, Thorkell Gudjónsson, Jan Benada, Jayashree Thatte, Jens Vilstrup Johansen, Judit Börcsök, Hanneke van der Gulden, Ji-Ying Song, Renée Menezes, Asma Tajik, Lucía Sena, Zoltan Szallasi, Morten Frödin, Jos Jonkers, Finn Cilius Nielsen, Claus Storgaard Sørensen

New Pseudomonas infections drive Pf phage transmission in CF airways

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Pf bacteriophages, lysogenic viruses that infect Pseudomonas aeruginosa (Pa), are implicated in the pathogenesis of chronic Pa infections; phage-infected (Pf+) strains are known to predominate in people with cystic fibrosis (pwCF) who are older and have more severe disease. However, the transmission patterns of Pf underlying the progressive dominance of Pf+ strains are unclear. In particular, it is unknown whether phage transmission commonly occurs horizontally between bacteria via viral particles within the airway or if Pf+ bacteria are mostly acquired via de novo Pseudomonas infections. Here, we studied Pa genomic sequences from 3 patient cohorts totaling 662 clinical isolates from 105 pwCF. We identified Pf+ isolates and analyzed transmission patterns of Pf within patients between genetically similar groups of bacteria called “clone types”. We found that Pf was predominantly passed down vertically within Pa clone types and rarely via horizontal transfer between clone types within the airway. Conversely, we found extensive evidence of Pa de novo infection by a new, genetically distinct Pf+ Pa. Finally, we observed that clinical isolates showed reduced activity of the type IV pilus and reduced susceptibility to Pf in vitro. These results cast new light on the transmission of virulence-associated phages in the clinical setting.

Authors

Julie D. Pourtois, Naomi L. Haddock, Aditi Gupta, Arya Khosravi, Hunter A. Martinez, Amelia K. Schmidt, Prema S. Prakash, Ronit Jain, Piper Fleming, Tony H. Chang, Carlos Milla, Patrick R. Secor, Giulio A. De Leo, Paul L. Bollyky, Elizabeth B. Burgener

Ceramide(d18:1/18:1)-Ndufa6 Interaction Inactivates Respiratory Complex I to Attenuate Oxidative-stress-driven Pathogenesis in Liver Ischemia/Reperfusion Injury

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Abstract

Oxidative stress driven by malfunctioning respiratory complex I (RC-I) is a crucial pathogenic factor in liver ischemia/reperfusion (I/R) injury. This study investigates the role of alkaline ceramidase 3 (ACER3) and its unsaturated long-chain ceramide (CER) substrates in regulating liver I/R injury through RC-I. Our findings demonstrated that I/R upregulated ACER3/Acer3 and decreased unsaturated long-chain CER levels in human and mouse livers. Both global and hepatocyte-specific Acer3 ablation, as well as treatment with CER(d18:1/18:1), led to a significant increase of CER(d18:1/18:1) levels in the liver, which mitigated the I/R-induced hepatocyte damage and inflammation in mice. Mechanistically, Acer3 modulated CER(d18:1/18:1) levels in mitochondria-associated membranes and endoplasmic reticulum (ER), thereby influencing the transport of CER(d18:1/18:1) from ER to mitochondria. Acer3 ablation and CER(d18:1/18:1) treatment elevated CER(d18:1/18:1) in mitochondria, where CER(d18:1/18:1) bound to the RC-I subunit Ndufa6 to inactivate RC-I and reduced reactive oxygen species production in the I/R-injured mouse liver. These findings underscore the role of CER(d18:1/18:1)-Ndufa6 interaction in suppressing RC-I-mediated oxidative-stress-driven pathogenesis in liver I/R Injury.

Authors

Kai Wang, Leyi Liao, Hanbiao Liang, Pengxiang Huang, Qingping Li, Baoxiong Zhuang, Chen Xie, Xiangyue Mo, Xuesong Deng, Jieyuan Li, Yang Lei, Minghui Zeng, Cungui Mao, Ruijuan Xu, Cuiting Liu, Xianqiu Wu, Jie Zhou, Biao Wang, Yiyi Li, Chuanjiang Li

Cellular immunophenotyping in human and primate tissues during healthy conditions and Ebola and Nipah infections

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Abstract

We developed a 29-color spectral cytometry panel to enhance nonhuman primate (NHP) models for cross-reactive immunophenotyping. This panel is suitable for biosafety level 4 (BSL-4) viruses and can be used with both human and NHP samples in BSL-2 research settings. Tissues from humans, rhesus monkeys (RhMs), crab-eating macaques (CEMs), and green monkeys (GMs) were stained with a 29-color immunophenotyping panel requiring only two clone substitutions. Comparable staining was observed for all samples. Unbiased analysis showed acceptable overlap in T-cell phenotypes across samples, with differences in human and NHP B cells and granulocytes. In CEMs, most circulating CD8+ T cells were from effector memory cells, with significantly higher levels than in humans (p<0.0001), RhMs (p<0.05), and GMs (p<0.01). Analysis of samples from various anatomical sites revealed distinct location-specific phenotypes. In Nipah-virus-exposed animals, splenocytes showed a substantial increase in IgM+ B cells (p<0.0001) and a reduction in effector memory CD8+ T cells (p<0.0001) compared to unexposed controls. Lymph nodes from Ebola-virus-exposed animals showed a loss of CXCR3+CD8+ T cells vs unexposed controls. This panel may guide the development of additional multi-color panels in preclinical and clinical settings and potentially increase understanding of the pathogenesis of diseases caused by emerging and re-emerging viruses.

Authors

Andrew P. Platt, Bobbi Barr, Anthony Marketon, Rebecca Bernbaum, Deja F.P. Rivera, Vincent J. Munster, Daniel S. Chertow, Michael R. Holbrook, Scott M. Anthony, Bapi Pahar

Multicomponent parenteral lipid emulsions do not prevent liver injury in neonatal pigs with obstructive cholestasis

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Abstract

Biliary atresia (BA) is a pediatric liver disease that often necessitates parenteral nutrition (PN) to support growth due to impaired liver function. While soy oil lipid emulsions (SLE) are commonly used in PN, they may contribute to cholestatic liver injury. In contrast, mixed oil lipid emulsions (MLE) show promise in preventing cholestasis in non-BA infants, potentially by restoring bile flow. However, their effectiveness in cases of complete bile duct obstruction, as seen in BA, remains uncertain. To explore the potential benefits of MLE in BA, we utilized a neonatal pig model of bile duct ligation (BDL). Pigs underwent either BDL or sham surgery and were subsequently fed either MLE or SLE via PN, or enterally with formula. The MLE-BDL pigs exhibited significantly greater weight gain compared to those fed SLE or formula enterally. Additionally, MLE-BDL pigs showed higher serum bile acid and gamma-glutamyl transferase concentrations compared to SLE-BDL pigs. However, no significant differences in liver injury, assessed by ductular reaction or fibrosis, were observed between MLE- and SLE-BDL pigs. Based on weight gain alone, MLE may be a superior lipid emulsion for use in neonates with obstructive cholestasis.

Authors

Greg Guthrie, Caitlin Vonderohe, Valeria Meléndez Hebib, Barbara Stoll, Douglas Burrin